Excessive airway mucus is an important cause of morbidity and mortality in asthma, but the relationship between accumulation of mucus and goblet cell size, number, and function is incompletely understood. To address these questions, stored mucin in the epithelium and goblet cell size and number were measured morphometrically, and mucin gene expression was measured by polymerase chain reaction and immunohistochemistry in endobronchial biopsies from 13 subjects with mild and moderate asthma and from 12 healthy control subjects. Secreted mucin was measured in induced sputum. We found that stored mucin in the airway epithelium was three times higher than normal in the subjects with asthma (p < 0.005). Goblet cell size was similar in both groups, but goblet cell number was significantly higher in the subjects with asthma (93,043 +/- 15,824 versus 41,959 +/- 9,230/mm3, p < 0.05). In mild asthma (FEV1 > or = 80% pred, n = 7), the level of stored mucin was as high as in moderate asthma (FEV1 < 80% pred, n = 6), but the level of secreted mucin was significantly lower (28.4 +/- 6.3 versus 73.5 +/- 47.5 microg/ml, p < 0.05). Secreted mucin was inversely correlated with stored mucin for the whole asthma group (rs = -0.78, p = 0.007). MUC5AC was the predominant mucin gene expressed in healthy subjects and subjects with asthma, and MUC5AC protein was increased in the subjects with asthma. We conclude that even mild asthma is associated with goblet cell hyperplasia and increased stored mucin in the airway epithelium, whereas moderate asthma is associated with increased stored mucin and secreted mucin. These findings suggest that acute degranulation of hyperplastic goblet cells may represent a mechanism for asthma exacerbations in mild and moderate asthma and that chronic degranulation of goblet cells may contribute to chronic airway narrowing in moderate asthma.

In smokers' lungs, excessive mucus clogs small airways, impairing respiration and promoting recurrent infection. A breakthrough in understanding this pathology was the realization that smoke could directly stimulate mucin synthesis in lung epithelial cells and that this phenomenon was dependent on the cell surface receptor for epidermal growth factor, EGFR. Distal steps in the smoke-triggered pathway have not yet been determined. We report here that the predominant airway mucin (MUC5AC) undergoes transcriptional up-regulation in response to tobacco smoke; this is mediated by an AP-1-containing response element, which binds JunD and Fra-2. These transcription factors require phosphorylation by upstream kinases JNK and ERK, respectively. Whereas ERK activation results from the upstream activation of EGFR, JNK activation is chiefly EGFR-independent. Our experiments demonstrated that smoke activates JNK via a Src-dependent, EGFRindependent signaling cascade initiated by smoke-induced reactive oxygen species. Taken together with our earlier results, these data indicate that the induction of mucin by smoke is the combined effect of mutually independent, reactive oxygen species activation of both EGFR and JNK.The primary cause of morbidity in chronic bronchitis is mucin overproduction, a phenomenon for which the molecular pathogenesis is unknown. Inflammatory cells are abundant in smokers' airways (1-3) and are capable of stimulating mucin production (4 -7), suggesting that at least some of the excessive mucin in smokers' lungs is secondary to inflammation.In addition, however, smoke itself can induce mucin synthesis in lung cells (8,9). The question of how this occurs is complex in that smoke, a composite of irritant molecules including acetaldehyde, hydroquinone, formaldehyde, benzo-[a]pyrene, cresol, nicotine, catechol, acrolein, coumarin, anthracene, nitrogen oxides, and heavy metals (10, 11) may act on lung epithelial cells in diverse ways. For example, the induction of cytochrome P450 by tobacco smoke (12) is mediated by binding of the aryl hydrocarbon nuclear receptor to a dioxin response element in the 5Ј-flank of the gene, but the induction of the ␥-glutamylcysteine synthetase heavy subunit (␥-GCS-HS) gene is mediated by the binding of a c-Jun/c-Jun homodimer to an AP-1-like response element (13).Previous reports have implicated the receptor for epidermal growth factor (EGFR) 1 in the induction of mucin gene MUC5AC by smoke (9). Consistent with a role for EGFR in mucin induction, an EGF response element has been identified 200 bp upstream of the MUC5AC gene (14). The response of this element to EGFR ligands EGF and transforming growth factor-␣ is mediated by Sp1. One might predict from these data that the induction of MUC5AC by smoke would depend on interaction between the EGF response element at Ϫ200 bp and Sp1.In contrast, in the present study we show that MUC5AC is controlled principally by a smoke response element ϳ3 kb upstream of the EGF response element. This element is AP-1-dependent and is bound by Ju...

Airway inflammation induced by gram-negative bacteria is often characterized by an influx of neutrophils and hypersecretion of mucus. The purpose of this study was to determine how endotoxin, a component of gram-negative bacteria and a chemotaxinogen for neutrophils, affects the amount of stored intraepithelial mucosubstances in the rat nasal airway. Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls). Before the first and third instillation, half of the animals were depleted of circulating blood neutrophils by administering a rabbit anti-rat neutrophil antiserum. Rats were sacrificed 6 or 24 h after the last instillation. Nasal tissues were processed for light microscopy and histochemical detection of stored intraepithelial mucosubstances. The numbers of nasal epithelial cells and intraepithelial neutrophils per millimeter of basal lamina in the anterior nasal septum and the amounts of intraepithelial mucosubstances in the same nasal tissue were determined by image analysis. We did not observe a neutrophil influx in the nasal tissues of neutrophil-depleted rats at 6 or 24 h after the last endotoxin instillation; however, we did observe a significant increase in intraepithelial mucosubstances, compared to saline-instilled controls. In contrast, nonneutrophil-depleted animals had a marked neutrophilic influx and a concomitant decrease in stored mucosubstances, compared to saline-instilled controls. There was no significant difference in the number of nasal epithelial cells per millimeter of basal lamina among any of the experimental groups. These results indicate (1) that endotoxin induces an increase in the amount of intraepithelial mucosubstances only when intraepithelial neutrophils are absent, and (2) that the endotoxin-induced, neutrophil influx probably triggers mucous hypersecretion.

Assessment of the acute systemic oral, dermal, and inhalation toxicities, skin and eye irritancy, and skin sensitisation potential of chemicals is required under regulatory schemes worldwide. In vivo studies conducted to assess these endpoints can sometimes be associated with substantial adverse effects in the test animals, and their use should always be scientifically justified. It has been argued that while information obtained from such acute tests provides data needed to meet classification and labelling regulations, it is of limited value for hazard and risk assessments. Inconsistent application of in vitro replacements, protocol requirements across regions, and bridging principles also contribute to unnecessary and redundant animal testing. Assessment of data from acute oral and dermal toxicity testing demonstrates that acute dermal testing rarely provides value for hazard assessment purposes when an acute oral study has been conducted. Options to waive requirements for acute oral and inhalation toxicity testing should be employed to avoid unnecessary in vivo studies. In vitro irritation models should receive wider adoption and be used to meet regulatory needs. Global requirements for sensitisation testing need continued harmonisation for both substance and mixture assessments. This paper highlights where alternative approaches or elimination of tests can reduce and refine animal use for acute toxicity requirements.

Tesfaigzi, Yohannes, J. Foster Harris, Jon A. Hotchkiss, and Jack R. Harkema. DNA synthesis and Bcl-2 expression during development of mucous cell metaplasia in airway epithelium of rats exposed to LPS. Am J Physiol Lung Cell Mol Physiol 286: L268-L274, 2004. First published October 3, 2003 10.1152/ ajplung.00172.2003.-Exposure of pulmonary airways to environmental toxins and allergens may cause proliferation of airway epithelial cells and mucous cell metaplasia (MCM); however, it is unclear to what extent proliferating cells differentiate into mucus-storing cells and contribute to MCM. Our previous studies demonstrated that Bcl-2, an inhibitor of apoptosis with cell cycle regulatory functions, is expressed in metaplastic mucous cells. The purpose of the present study was to investigate the number of metaplastic mucous cells that are derived from proliferating epithelial cells and whether Bcl-2 has a role in cell cycle entry in these cells. Rats were intratracheally instilled with 100 g of LPS from Pseudomonas aeruginosa in 500 l of saline, and proliferating airway cells were labeled with bromodeoxyuridine (BrdU) by implanting a subcutaneous osmotic pump 24 h before instillation. The volume of stored mucosubstance and the number of mucous cells were increased 10-and 3-fold, respectively, from 24-48 h after instillation. The number of total epithelial cells per millimeter of basal lamina increased, and the number of serous cells per millimeter of basal lamina decreased during this time. Approximately 50% of Alcian blue-periodic acid Schiff-stained mucous cells were labeled with BrdU at 48 h after instillation, suggesting that one-half of the secretory cells were derived from proliferating cells. Furthermore, 50% of the Bcl-2-positive mucous cells were BrdU negative and therefore derived from nonproliferating, preexisting cells. Our findings demonstrate that preexisting and proliferating cells differentiate into mucous cells and compose LPS-induced metaplasia and that Bcl-2 does not have cell cycle regulatory function in these cells.bromodeoxyuridine; secretory cell; apoptosis; electron microscopy EXPOSURE OF THE PULMONARY airways to inhaled toxins or allergens may cause injury to the airway epithelium that results in an acute inflammatory response, proliferation of surviving epithelial cells, and mucous cell metaplasia (MCM). MCM occurs in respiratory epithelia when nonproliferating, preexisting or proliferating epithelial cells differentiate into mucous cells in areas where no or few mucous cells are normally present. Increased numbers of airway mucous cells is a prominent feature of a number of bronchial diseases in humans, including chronic bronchitis (13), asthma (24), and cystic fibrosis (28). Proliferation of epithelial cells was reported in airways of subjects with chronic bronchitis (4); however, it is unknown to what extent existing or proliferating cells differentiate into mucus-storing cells and contribute to the development of MCM. Exposure to LPS (endotoxin) from the outer membrane of gram-negative ...